Methods and nucleotide fragments of predicting occurrence, metastasis of cancers and patients&#39; postoperative survival in vitro

ABSTRACT

Provided in the present invention are a method using in vitro measurement of the content of methylation or demethylation of GFRa1 CpG islands to estimate a risk of tumorigenesis and of tumor metastasis, or postoperative life expectancy, and a nucleotide sequence used.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No,14/378,285, filed Aug. 12, 2017, which is a nationalization under 35U.S.C. § 371 from International Application Serial No.PCT/CN2012/000169, filed Feb. 13, 2012 and published as WO2013/120222 onAug. 22, 2013, the contents of which application and publication areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to methods of predicting the ability ofmalignant tumor invasion, metastasis in vitro and length of patients'survival time, and also relates to the nucleotide fragments used in themethods.

TECHNICAL BACKGROUND

Invasion and metastasis are predominantly reasons for the poor prognosisof cancers. Destructions of the neighbor and distant organs by cancerinvasion and metastasis lead to loss of chance for surgical resectionand recurrence after curative treatments. Sensitive biomarkers fordetection of potential of invasion and metastasis would greatly improvethe personalized clinical management for cancer patients. Therefore,predicting the invasion and metastasis potential of cancers is eagerlyawaited.

It is well recognized that it is virtually impossible to identifymetastasis potential of cancers based on histopathologic grounds alone.So it is expected to make molecular subtyping using the molecularbiology methods. Great progress has been achieved on the expressionchange of protein and RNA in the past decades. Although there are manystudies on cancer biology, the effectual method is still unavailable toaccurately recognize the metastasis ability of cancer cells.

As the rapid development of molecular biology, people have got acomprehensive understanding on the mechanisms of carcinogenesis. Inaddition to the genetic inactivation or activation of tumor relatedgenes (including p53, APC and Ras, etc.) epigenetic inactivation oftumor suppressor genes (including p15, p16 and hMLH1, etc.) byhypermethylation and reactivation of proto-oncogenes by hypomethylationof CpG islands are other kinds of frequent events in cancers. It is wellknown that detection of alterations of protein levels and mRNA levels ofgenes in a few abnormal cells in tissue samples is very difficult usingregular gene expression assays, because their visibility would begreatly reduced by the co-existence of main cell populations in whichthe gene expression has not changed. In contrast, methylated anddemethylated CpG islands can be analyzed with methylation- anddemethylation-specific assays, respectively. This makes the detection ofthe methylation status of CpG islands so sensitive that methylationalterations that occurred in a few cells in a testing tissue can beclearly displayed. This makes DNA methylation an optimal biomarker formolecular stratification of cancers.

Receptor GFRa1 combines with Glial cell line-Derived Neurotrophic Factor(GDNF), forming the phosphotyrosine kinase [Cell 1996, 85(7):1113-1124]of the oncogene RET which is capable of activating the signalingpathways such as SRC, MAPK. AKT and Rho, etc. It is closely related tothe proliferation, differentiation and migration of the cells [NatureReviews Neuroscience 2002, 3(5):383-394]. It has been discovered thatGFRa1 expression is elevated in the tissues of a number of cancers (suchas pancreatic cancer, breast cancer, olfactory cell carcinoma and theglial cell tumor). Elevated expression of this gene promotes theoccurrence, development and metastasis of these cancers [Cancer Research2005, 65(24):11536-11541; Cancer Research 2007, 67(24): 11733-11741]. Ithas also been reported that the methylation-deactivation of GDNF, theligand of GFRa1, is related to the occurrence of gastric cancer[Gastroenterology 2009; 136:2149-2158]. But there is no report on themethod of using the methylation and demethylation of GFRa1 CpG islandsto estimate the occurrence, metastasis and survival of tumors.

DETAILED DESCRIPTION OF INVENTION

On one hand, the present invention provides an in vitro detection assayfor the occurrence, metastasis, and survival time of tumor and theartificial nucleotide used in the method. It will help with the earlydiscovery and definite diagnosis making of tumor, the accurateestimation of the metastasis ability of tumor and the postoperativesurvival time of patients to provide help for the diagnosis andtreatment of tumor.

In order to obtain the above effectives, the invention provides thefollowing technical proposal.

An in vitro detection assay for occurrence of tumor is disclosed in theinvention, which including the following steps:

-   -   a) Extracting of genomic DNA or plasma free DNA samples from        cancer patients and normal patients respectively;    -   b) Detecting and calculating proportion of methylation (or        demethylation) of GFRa1 CpG islands, determinating of a cutoff        value of methylation (or demethylation) for tumor,    -   c) Extracting of genomic DNA or plasma free DNA from testing        patient and detecting and calculating of methylation (or        demethylation) proportion of GFRa1 CpG islands;    -   d) Comparing the methylation (or demethylation) proportion        determined in the step c) with the cutoff value of methylation        (or demethylation) determined in the step b).    -   e) If the methylation (or demethylation) proportion determined        in the step c) is less (or greater) than or equal to the cutoff        value of methylation (or demethylation) determined in the step        b), occurrence of tumor should be considered; or    -   If the methylation (or demethylation) proportion in step c) is        greater (or less) than the cutoff value of methylation (or        demethylation) determined in the step b), occurrence of tumor is        not considered.

Further, the methods of detection and calculation of the GFRa1 CpGislands methylation (or demethylation) proportion in step b) and c) areas follows: chemical modifying of the unmethylated cytosine; designationand synthesis of PCR primers which can match with the methylated (ordemethylated) CpG island of modified GFRa1 sequences; amplification ofmethylated (or demethylated) GFRa1 CpG islands using these primers;detection and calculation of the methylation (or demethylation)proportion of GFRa1 CpG islands using quantitative methylation assays.Other methods in the art can also be used to analyze the GFRa1methylation level, such as sequencing based on methylated DNA enrichmentand the combination with other technologies.

Further, the cutoff value of methylation (or demethylation) proportionof GFRa1 CpG islands is determined through the method of ROC curve instep b).

The invention further involves the modified DNA sequences of GFRa1 CpGislands mentioned in step b) and step c) are shown in SEQ ID NO. 1, SEQID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

Further more, the GFRa1 methylation (or demethylation) proportion instep b) and step c) is quantified with DHPLC, bisulfite-sequencing, orpro-based, quantitative, methylation-specific PCR (MethyLight).

Preferably, the primer sets in the quantitative methylation analysisused DHPLC or bisulfite-sequencing are:

-   -   a) The primer set whose base sequences are showed in SEQ ID NO.5        and SEQ ID NO.6; or    -   b) The primer set whose base sequences are showed in SEQ ID NO.7        and SEQ ID NO.8.

Preferably, the primer sets and probes in the MethyLight analysis are:

-   -   a) The oligonucleotide group that is made up of the primer set        whose base sequences are showed in SEQ ID NO.9 and SEQ ID NO.10,        and the probe whose base sequence is showed in SEQ ID NO. 11, or    -   b) The oligonucleotide group which is made up the primer set        whose base sequences are showed in SEQ ID NO.12 and SEQ ID        NO.13, and the probe whose base sequence is showed in SEQ ID        NO.14.

Further, the tumors mentioned in this invention are selected from coloncancer, gastric cancer or liver cancer.

This invention also provides a method of in vitro detection assay forrisk of metastasis of cancer and postoperative survival time methodincluding the following steps:

-   -   a) Extraction of DNA from the tumor tissues of patients with        metastatic cancer and the tumor tissues of patients with        non-metastatic cancer,    -   b) Detection and calculation of methylation (or demethylation)        proportion of GFRa1 CpG islands and determination of the cutoff        value for prediction of cancer metastasis.    -   c) Extraction of tumor DNA of testing cancer patient, detection        and calculation of methylation (or demethylation) proportion of        GFRa1 CpG island in the tumor tissue.    -   d) Comparison between the GFRa1 methylation (or demethylation)        proportion determined in the step c) and the methylation (or        demethylation) cutoff value determined in the step b).    -   e) If the methylation (or demethylation) proportion determined        in the step c) is less (or greater) than or equal to the cutoff        value determined in the step b), it will be estimated that the        patient has a high risk of tumor metastasis and a short        postoperative survival; or        -   If the methylation (or demethylation) proportion determined            in the step c) is greater (or less) than the cutoff value            determined in the step b), it will be estimated that the            patient has low risk of tumor metastasis and a long            postoperative survival. On the contrary, it will be            estimated that the patient has a high risk of tumor            metastasis and a short postoperative survival.

Further, the method of detecting and calculating the methylation (ordemethylation) proportion determined in the step b) and the step c) isas follows: chemical modification of the unmethylated cytosine;designation and synthesis of PCR primers which can match with themethylated (or demethylated) CpG island of modified GFRa1 sequences;amplification of methylated (or demethylated) GFRa1 CpG islands usingthese primers; detection and calculation of the methylation (ordemethylation) proportion of GFRa1 CpG islands using quantitativemethylation assays. Other methods can also be used to analyze the GFRa1methylation level, such as sequencing based on methylated DNA enrichmentand the combination with other technologies.

Further, the cutoff value of methylated (or demethylated) GFRa1proportion calculated in the step b) is determined through the method ofROC curve.

Further, the oligo sequence of the modified sequence of GFRa1 CpG islandin step b) and c) is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3or SEQ ID NO.4.

Further, quantitative analysis of GFRa1 methylation content in the stepsb) and c) is carried out with DHPLC, bisulfite-sequencing, andMethyLight (probe-based, quantitative, methylation-specific PCR).

Further, in the quantitative analysis of methylation in the DHPLC andbisulfite-sequencing assay, the primer set mentioned in this inventionis:

-   -   a) The primer set that is made up of oligonucleotides whose base        sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6; or    -   b) The primer set that is made up of oligonucleotides whose base        sequence is as shown in SEQ ID NO.7 and SEQ ID NO.8.

Further, in the method of quantitative analysis of methylation inMethyLight assay, the oligonucleotide group mentioned in this inventionis:

-   -   a) The oligonucleotide group that is made up of primer set whose        base sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10; and        the probe whose base sequence is as shown in SEQ ID NO.11; or    -   b) The oligonucleotide group which is made up of primer set        whose base sequence is as shown in SEQ ID NO.12 and SEQ ID        NO.13, and the probe whose base sequence is as shown in SEQ ID        NO.14.

Further, the tumor mentioned in this invention refers to stomach, colonor liver cancers.

This invention also provides a kind of DNA molecule whose base sequenceis as showed in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO. 4.

This invention also provides a kind of primer set, whose base sequenceis as showed in SEQ ID NO.5 and SEQ ID NO.6.

This invention also provides another kind of primer set, whose basesequence is as showed in SEQ ID NO.7 and SEQ ID NO.8.

This invention also provides a kind of oligonucleotide group, whichincludes primer set whose base sequence is as showed in SEQ ID NO.9 andSEQ ID NO.10 and the probe whose base sequence is as showed in SEQ IDNO.11.

This invention also provides a kind of oligonucleotide group, whichincludes primer set whose base sequence is as showed in SEQ ID NO.12 andSEQ ID NO.13, and the probe whose base sequence is as showed in SEQ IDNO.14.

This invention also provides a method to detect abnormal reactivation ofGFRa1 expression. The feature of the method is: analysis of fulldemethylation of CpG sites around the transcription start site of GFRa1gene. This is first recognized in the world by the inventors of theinvention. Further, the invention provides a set of assays to detectabnormal reactivation of GFRa1 for determination of occurrence andmetastasis of cancers and patient's postoperative survival.

In order to achieve the goals above, these methods are put forwardedbased on the following research results.

Hypothesis

The occurrence and progression of tumor is a multi-factor, multi-pathwayand multi-stage process. Epigenetic signatures and biological featuresof cancer cells may be pathway-dependent, which will lead to differentprognosis. For example, the prognosis of colorectal cancer withmicrosatellite instability-high (MSI-H, related to inactivation ofmismatch-repair genes such as MLH1) is better than those with MSI-low;prognosis of gliomas with DNA repair gene MGMT inactivation bymethylation is better than those without MGMT methylation. There is aCpG island around the transcription start site of GFRa1 gene. GFRa1 isinactivated by methylation in most normal adult tissues. However itsexpression is obviously upregulated in many cancers. GFRa1 promotes theoccurrence and progression of cancers. The inventors have firstly foundthat GFRa1 is demethylated in cancer tissues and the demethylationsubsequently leads to upregulation of GFRa1 in cancers. According tothese, the inventors hypothesize that detection of methylated (ordemethylated) GFRa1 content could be used to determine occurrence ofcancer, to predict metastasis of cancers and patients' survival. Thishypothesis has been validated in many cancers as described below.

Validation

-   1. Clue: Our genome-wide DNA methylome data shows that methylation    signal ratio [gastric cancer (GC) vs. surgical margin (SM)] of GFRa1    promoter detected with microarray probes is significantly lower in    the metastatic GCs than the non-metastatic GCs (FIG. 1). This    suggests that GFRa1 reactivation by DNA demethylation may be a    potential tumor biomarker. Therefore, following experiments are    carried out to confirm this hypothesis in details.-   2. Establishment of methods to quantitatively detect GFRa1    methylation and demethylation: the unmethylated cytosine residues in    genomic DNA samples are converted to uracil residues with sodium    bisulfite. A CpG-free primer set is used to amplify both the    methylated and unmethylated target DNA fragments (522 bp) of GFRa1    CpG islands. Denaturing high-performance liquid chromatography    (DHPLC) is used to separate and quantify the amounts of methylated    and unmethylated GFRa1 molecules in the PCR products (FIG. 2).    Results of bisulfite clone sequencing, a semi-quantitative assay,    are consistent with DHPLC analysis (FIG. 3). These indicate that    DHPLC assay could be used to quantitative analysis of ratio of    methylated to unmethylated GFRa1 alleles. In order to detect GFRa1    methylation in DNA samples from the paraffin-embedded tissues, in    which high-molecule DNA are broken into small-molecule DNA, a    probe-based, quantitative, methylation-specific PCR assay    (MethyLight) is setup to determine the methylation level in a 158 bp    fragment within the core CpG island of the gene. After normalized    for input DNA using the CpG-free region in reference gene COL2A1    (Widschwendter et al. Cancer Res 2004, 64:3807-3813), results of the    MethyLight analysis are highly consistent with DHPLC analysis    (r=0.5; P=0.000; FIG. 4). Similar association between GFRa1    methylation and cancer characteristics could be observed in both    DHPLC and MethyLight analysis.-   3. Reactivation of GFRa1 expression by demethylation of CpG islands:    In cell lines and tissue samples with different GFRa1 methylation    states, the mRNA level of GFRa1 gene is analyzed with a    fluorescence-probe based, quantitative RT-PCR. Results shows that    GFRa1 mRNA is detected in 4 GFRa1 demethylation positive cell lines,    but not in 15 demethylation negative cell lines (P<0.001; Figure    SA). Similarly, it is observed that GFRa1 mRNA levels are inversely    correlated with GFRa1 methylation levels in gastric tissue samples    (P=0.041; FIG. 5B). The same phenomenon is also observed in the    colon tissues. To sum up, these results indicate that promoter DNA    demethylation may reactivate GFRa1 expression.-   4. GFRa1 demethylation in gastric tissues is a potential biomarker    for screen of gastric cancer: GFRa1 demethylation levels in    normal/gastritis biopsies from 48 non-cancer control patients (10    normal and 38 chronic gastritis), 98 gastric cancers and the    corresponding surgical margin “normal” tissue samples are analyzed    by using of DHPLC. The results show that the methylated:    demethylated GFRa1 ratio in the normal/gastritis biopsies (Median,    60.4%) is significantly higher than that in GCs (51.0%, P=0.043) or    SM samples (14.5%; P<0.01). It indicates that GFRa1 is demethylated    in the development of gastric cancer and that the demethylation    occurs both in gastric cancer tissues and adjacent non-cancerous    tissues as a field effect. Therefore, it is very useful for    screening of gastric cancer at early stage using gastric biopsies,    in which cancer cells may not be sampled in some cases.

To investigate the feasibility of screening of gastric carcinomas usingGFRa1 as a biomarker, the cutoff value of GFRa1 methylation:demethylation ratio is calculated using the receiver operatingcharacteristic curve (ROC). The area under the ROC is 67.3% (P<0.001,FIG. 6B), according to above GFRa1 methylation results for gastriccancer and non-cancer patients. When the cutoff value is set at 22.8%(≤22.8% for demethylation positive and >22.8% for demethylationnegative), the demethylation positive rate in normal/gastritis samples(15/48=31.2%) is much lower than the surgical margin (77/98=78.5%) andgastric cancer tissues (60/98=61.2%) (P<0.001). Sensitivity andspecificity of GFRa1 demethylation positive in surgical margin tissuefor screening of gastric cancer is 79% and 69%, respectively.

-   5. GFRa1 demethylation is a potential biomarker for prediction    metastasis of gastric carcinomas and patients' overall survival: In    analysis of relationship between GFRa1 demethylation and gastric    cancer metastasis or patients' overall survival, it is found that    the proportion of methylated-GFRa1 in 49 non-metastatic gastric    carcinomas is significantly higher than that in 49 metastatic    carcinomas (Median, 60.6% vs. 22.8%, P=0.044). Therefore, the    metastasis status of gastric carcinoma is used as a golden standard    to calculate the ROC curve to evaluate the efficiency to use GFRa1    methylation as a classifier for prediction of cancer metastasis. It    is found the area under the ROC curve (AUC) is 65.6% (P=0.004; FIG.    7). When the cutoff value of the proportion of methylated-GFRa1 is    set at 16.4% (≤16.4% for GFRa1 demethylation-high and >16.4% for the    demethylation-low), the demethylation-high rate in metastatic    gastric carcinomas 71% (35/49) is significantly higher than in    non-metastatic gastric carcinomas 49% (24/49; P=0.038) (sensitivity    of 71% and specificity of 51%). Kaplan-Meier analysis shows that    GFRa1 demethylation-high patients' overall survival is shorter than    the demethylation-low patients (5-year survival rate, 32.8% vs. 62%;    log-rank test, P=0.001; multivariate analysis, P=0.002; FIG. 8).

Above findings are further validated among 120 independent patients withgastric carcinomas without distant metastasis. Again, it is found thatthe proportion of methylated-GFRa1 in 47 non-metastatic cancer tissuesis significantly higher than that in 73 lymphonodus metastatic cases(Median. 49.0% vs. 30.6%, P<0.001). Using the same cutoff value (16.4%),GFRa1 demethylation-high rate in the metastatic cases (46/73) issignificantly higher than that in non-metastatic cases (19/47) (63.1%vs. 40.4%, P=0.024; sensitivity of 63% and specificity of 60%).Kaplan-Meier analysis also shows that the demethylation-high patientshave a significant shorter overall survival than demethylation-lowpatients (5-year survival rate, 47.7% vs. 71.7%; log-rank test, P=0.015;multivariate analysis, P=0.025; FIG. 9). Results in the discovery andvalidation studies show that demethylation levels of GFRa1 CpG islandsin gastric carcinomas are closely correlated with cancer metastasis andoverall survival of patients. These results indicate GFRa1 demethylationmay be a useful biomarker for prediction of cancer metastasis.

-   6. GFRa1 demethylation is a biomarker to predict occurrence and    metastasis of colon cancer and patients' survival. In order to study    relationship between GFRa1 demethylation and prognosis of other    cancers, GFRa1 demethylation levels in colon mucosal biopsy samples    from 21 non-cancer patients, 97 colon cancers and their    corresponding surgical margin tissues are analyzed with the DHPLC    assay. The results show that the proportion of methylated-GFRa1 in    the colon biopsies (Median, 64.1%) is significantly higher than    colon carcinomas (31.6%; P=0.001) and the surgical margin tissues    (26.6%; P<0.001; FIG. 10). The area under the ROC curve is 74.1%    according to results of the methylation analysis using cancer    tissues and control biopsies from non-cancer patients (P<0.001; FIG.    11). When the cutoff value is set at 34.5% (≤34.5% for demethylation    positive and >34.5% for demethylation negative), GFRa1 demethylation    positive rate in 7/21 control biopsies from non-cancer patients    (33.3%) is significantly lower than that in 93/97 surgical margin    sample (95.8%; P<0.001) and 60/97 cancer samples (61.9%; P<0.001).    Using GFRa1 demethylation positive in the non-cancer biopsies or    surgical margin samples as a biomarker for screening of colon    cancer, the sensitivity and specificity is 95.8% and 66.7%,    respectively.

Furthermore, relationship between GFRa1 demethylation and colon cancermetastasis is also analyzed among these 97 colon cancer patients (49non-metastatic and 48 metastatic cancers). The proportion ofmethylated-GFRa1 in 49 non-metastasis cases is significantly higher thanthat in 48 metastasis cases (Median, 45.6% vs. 25.0%, P=0.016). The areaunder the ROC curve (AUC) is 62.6% (P=0.033; FIG. 12). When the cutoffvalue is set at 27.6% (≤27.6% for GFRa1 demethylation-high and >27.6%for the demethylation-low), the GFRa1 demethylation-high positive ratein 48 metastatic cancers (33/48=68.8%) is significant higher than in 49non-metastasis cancers (22/49=44.9%; P=0.024; sensitivity of 69% andspecificity of 55%). Kaplan-Meier analysis showed that GFRa1demethylation-high cancer patients had a significant shorter overallsurvival than demethylation-low patients (3-year survival rate, 41.8%vs. 66.7%; log-rank test, P=0.019; multivariate analysis, P=0.031; FIG.13).

Similar results are observed when the methylation-specific fluorescencequantitative PCR is used to detect the GFRa1 demethylation level inthese tissues. The area under the ROC curve (AUC) is 61.7% (P<0.05; FIG.14). When the cutoff value is set at 22.3% (≤22.3% for GFRa1demethylation-high and >22.3% for the demethylation-low), the GFRa1demethylation-high positive rate in 48 metastatic cancers (40/48) issignificant higher than in 49 non-metastasis cancers (31/49) (83.3% vs.63.3%, P=0.038; sensitivity of 83% and specificity of 37%). Kaplan-Meieranalysis shows that GFRa1 demethylation-high cancer patients have asignificant shorter overall survival than demethylation-low patients(3-year survival rate, 46.5% vs. 69.2%; FIG. 15).

-   7. GFRa1 demethylation is a biomarker to predict occurrence and    metastasis of other cancers and patients' survival. Relationship    between GFRa1 demethylation and prognosis of hepatocellular    carcinomas (HCCs) is also analyzed. Results shows that the    proportion of methylated GFRa1 alleles in 20 non-metastatic HCCs is    higher than that in 17 metastatic HCCs (Median, 55.6% vs 41.4%,    P=0.065). The area under the ROC curve (AUC) is 68.4% (FIG. 16).    When the cutoff value is set at 49.3% (≤49.3% for GFRa1    demethylation-high and >49.3% for the demethylation-low), GFRa1    demethylation-high positive rate in 17 metastatic HCCs (12/17) is    significantly higher than in 20 non-metastatic HCCs (11/20) (70.5%    vs. 55%, P=0.024, sensitivity of 70% and specificity of 45%).    Kaplan-Meier analysis also shows that the GFRa1 demethylation-high    patients have a shorter overall survival than the GFRa1    demethylation-low patients (FIG. 17).

In addition, it is found that GFRa1 CpG islands are completelydemethylated in lung cancer cell A549 and prostate cancer cells PC-3.The results described above indicate that GFRa1 demethylation isassociated with occurrence of multiple cancers.

To sum up, the present data shows that demethylation of GFRa1 CpGislands is a potential biomarker for screening of various cancers andhigh level of GFRa1 demethylation can be used to predict metastasis ofmultiple cancers and patients' overall survival time.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 refers to comparison of intensity of DNA methylation signal atdifferent sectors in GFRa1 CpG islands in the database of DNA methylomeof gastric carcinomas and the surgical margin tissues. FIG. 1A islocations of the 522 bp amplicon (the black thick lines) and microarrayprobes for detection of methylation (the black squares) for GFRa1 CpGisland. FIG. 1B is comparison of intensity of DNA methylation signal atdifferent sectors of GFRa1 CpG islands in the DNA methylome database.Differential methylation in the promoter region is observed betweenmetastatic gastric carcinoma (M+) and non-metastatic gastric cancer(M−).

FIG. 2 is chromatograms of 522 bp amplicon of methylated anddemethylated GFRa1 CpG islands in the bisulfite-DHPLC analysis. FIG. 2Arefers to result of demethylated GFRa1 of human cell lines; FIG. 2B isfor gastric tissue; Genomic DNA of peripheral white blood cells is usedas the negative control; M.sssI-methylated blood DNA is used as thepositive control.

FIG. 3 is results of bisulfite-sequencing of the 522 bp PCR clones fromGFRa1 CpG islands in human cell lines. The strong black dots representmethylated CpG sites; GFRa1 is fully methylated in Du145, MKN45 andBGC823 cells, partly methylated in MKN74 cell, and demethylated in GES-1cell.

FIG. 4 is the correlation analysis of GFRa1 methylation levels detectedwith DHPLC and fluorescence-probe-based, quantitative,methylation-specific PCR (MethyLight) analysis.

FIG. 5 shows relationship between GFRa1 demethylation and GFRa1transcription level. FIG. 5A is correlation analysis using 19 human celllines; FIG. 5B is correlation analysis using gastric carcinomas and thesurgical margin tissues.

FIG. 6 indicates the comparison of GFRa1 methylation level betweennormal/or gastritis biopsies from non-cancer patients and gastriccarcinomas and the surgical margin samples. FIG. 6A shows percentages ofmethylated GFRa1 in gastric tissues at different pathological states;FIG. 6B shows the cutoff value of ROC curve of detection of gastriccarcinoma using GFRa1 methylation content in the gastric tissues fromcancer and non-cancer patients.

FIG. 7 is the ROC curve of detection of gastric cancer metastasis byusing GFRa1 demethylation content as a classifier.

FIG. 8 shows the Kaplan-Meier curve of post-operational overall survivaltime of 98 GFRa1 demethylation-high or demethylation-low gastric cancerpatients in the discovery cohort.

FIG. 9 shows the Kaplan-Meier curve of post-operational overall survivalof 120 GFRa1 demethylation-high or demethylation-low gastric cancerpatients in the validation cohort.

FIG. 10 is the comparison of GFRa1 methylation level between colonbiopsies from non-cancer patients, colon cancer and the surgical marginsamples.

FIG. 11 is the cutoff value of ROC curve of detection of colon carcinomausing GFRa1 methylation content in colon biopsies from non-cancerpatients and the surgical margin tissue samples from colon cancerpatients.

FIG. 12 is the ROC curve of detection of metastasis and non-metastasisof colon cancers using the extent of GFRa1 demethylation.

FIG. 13 is the Kaplan-Meier curve of post-operational overall survivaltime of 97 GFRa1 demethylation-high or demethylation-low colon cancerpatients.

FIG. 14 is the ROC curve of detection of metastasis and non-metastasisof colon cancers using GFRa1 demethylation-high or demethylation-low,determined by using a probe-based, quantitative, methylation-specificPCR (MethyLight) assay, as a classifier.

FIG. 15 is the Kaplan-Meier curve of post-operational overall survivaltime of 97 patients with colon cancers containing GFRa1demethylation-high or demethylation-low in the probe-based,quantitative, methylation-specific PCR (MethyLight) analysis.

FIG. 16 is the ROC curve of detection of metastasis and non-metastasisof HCCs by using GFRa1 demethylation-high or demethylation-low.

FIG. 17 is the Kaplan-Meier curve of post-operational overall survivaltime of 37 patients with different GFRa1 demethylationextent.

FIG. 18 is the results of bisulfite-sequencing of 522 bp PCR products ofGFRa1 CpG islands in gastric tissue samples. The strong black dotsrepresent methylated CpG sites; GFRa1 is completely demethylated insamples F1309 and F1311, but not demethylated in other representativesamples.

Next, the detailed illustration of the invention will be made throughliving example. If it is not pointed out, the materials, methods andequipment are all the regular materials, methods and equipment in thisarea.

BEST EMBODIMENTS EXAMPLE 1 Screening for Gastric Cancer ThroughDetection of Demethylation of GFRa1 CpG Islands in Gastric Tissue byUsing of DHPLC

1. Subjects: gastric mucosa biopsies from 48 non-cancer patients (10cases without observed pathological changes in the stomach and 38 caseswith chronic gastritis), gastric carcinomas and the paired surgicalmargin frozen tissue samples from 98 patients;

2. Regularly digest tissue protein using proteinase K and thenextracting genomic DNA (about 10 μg) using the regular ethanolprecipitation method.

3. Modifying the unmethylated cytosine residues in DNA samples using SMsodium bisulfite, including following steps:

-   -   1) Adding and dissolving 2 μg genomic DNA prepared in the step 2        into 18 μl of distilled sterile water, incubating the tube in        water bath at 95° C. for 20 min, and then incubating in the ice        bath.    -   2) Adding 2 μl of 3M NaOH, mixing and incubating at 42° C. for        20 min to denature double strands DNA.    -   3) Preparing for SM sodium bisulfite solution (NaHSO₃, 4 ml):        adding 1.9 g of Na₂S₂O₅ into 2.5 ml distilled sterile water,        adding 0.7 ml of 2M NaOH solution and 0.5 ml of 1M hydroquinone,        incubating in a water bath at 50° C., with repeatedly inverting        and mixing until completely dissolved.    -   4) Adding 380 μl of fresh 5M NaHSO₃ solution and mixing.        Covering the top of reaction with 200 μl of liquid paraffin to        prevent evaporation of reaction. Incubating in the water bath at        50° C. overnight to convert the unmethylated cytosine residues        to uracil residues.    -   5) Removing liquid paraffin. Purifying the modified DNA with        Wizard DNA Clean-Up System (Promega A7280) as the kit        instruction: adding 1 ml of the mixture of resin and staying for        5 minutes after mixing; transferring the resin-DNA mixture into        an injector tube connected to a micro-column filter, removing        liquid phase and transferring the DNA sample onto solid phase in        the micro-column filter through vacuum; adding 2 ml of 80%        isopropyl alcohol, removing the liquid within the        injector-filter through vacuum, and disconnecting the injector        tube; setting the micro-column filter into another centrifuge        tube (1.5 ml), centrifuging at high-speed (10,000 g, 20 seconds)        to remove residual liquid in the column filter; setting the        micro-column filter to another centrifuge tube.    -   6) Adding 50 μl of distilled sterile water (pre-warmed at 8° C.)        into the micro-column, standing for 15 min, centrifuging at        high-speed (10,000 g, 20 seconds) to collect the eluent.        Repeating this washing process again. Pooling the eluent        solution.    -   7) Adding 11 μl of 3M NaOH solution, mixing and incubating in        the water bath at 37° C. for 15 min to terminate further        modification.    -   8) Adding 166 μl of 5M NaOAC and 750 μl of 100% cold ethanol,        mixing and storing at −20° C. for 4 hours to precipitate the        DNA. Centrifuging at 10,000 g for 30 min and discarding the        solution. Adding 200 μl of 80% cold ethanol to wash the DNA.        Centrifuging again and discarding the solution.    -   9) Resuspending the DNA in 3˜6 μl of sterile water or TE buffer.        Using immediately or storing at −20° C.

4. Design of PCR primer sets. According to the modified GFRa1sense-strand sequence (SEQ ID NO:1 and SEQ ID NO:2), designing andsynthesizing CpG-free universal primer sets (SEQ ID NO:5; the SEQ IDNO:6 or SEQ ID NO:7; the SEQ ID NO:8).

5. PCR amplification. Both the methylated and demethylated fragments(522 bp or 463 bp) in GFRa1 alleles in the modified DNA sample areamplified by using a hot start PCR.

6. Detection of the methylated and demethylated GFRa1 CpG islands in thePCR product by using of DHPLC. Calculating the peak-area proportion forthe demethylated GFRa1:

-   -   the percentage of demethylated GRFa1=[the peak area for        demethylated GFRa1]/[total peak areas for both demethylated and        methylated GFRa1PCR products]×100%;    -   or calculating the methylated GFRa1:    -   the percentage of methylated GRFa1=[the peak area for methylated        GFRa1]/[total peak areas for both demethylated and methylated        GFRa1 PCR products]×100%, or,    -   the percentage of methylated GRFa1=1−(the peak area proportion        for demethylated GFRa1).

7. Result: The average peak-area proportion for the methylated GFRa1 innormal gastric biopsies is similar to that in gastritis biopsies and theaverage percentage of methylated GFRa1 in these normal/gastritis samplesis 60.4% (Median), which is significantly higher than that in gastriccarcinomas and the surgical margin samples (51.0%, P=0.043; and 14.5%,P=0.000; FIG. 6A), respectively. Therefore, the receiver operatingcharacteristic curve (ROC) for screening of gastric carcinomas usingGFRa1 methylation percentage as a biomarker is calculated. When thecutoff value is set at 22.8% (≤22.8% for demethylation positiveand >22.8% for demethylation negative), the demethylation positive ratein normal/gastritis samples (15/48=31.2%) is much lower than thesurgical margin (77/98=78.5%) and gastric cancer tissues (60/98=61.2%)(P<0.001). Sensitivity and specificity of GFRa1 demethylation positivein surgical margin tissue for screening of gastric cancer is 79% and69%, respectively (FIG. 2B).

EXAMPLE 2 Detection of Metastasis of Gastric Carcinomas andPatients'Survival Through Detection of Demethylation of GFRa1 CpGIslands by Using of DHPLC

1. Subjects: gastric carcinoma frozen-tissue samples from 98 patients(including 49 patients with gastric carcinomas with metastasis to lymphor distant sites and embolus and 49 patients with non-metastatic gastriccarcinomas) in the discovery cohort. Gastric carcinoma frozen-tissuesamples from 120 patients (including 73 gastric carcinomas with lymphbut not distant metastasis and 47 non-metastatic gastric carcinomas) inthe validation cohort. Clinicopathologic and follow-up data areavailable for all of these subjects.

2. The same as the steps 2-5 in the Example 1;

3. The same as the step 6 in the Example 1;

4. Result:

The average peak-area proportion for the methylated GFRa1 in 49non-metastatic gastric carcinoma samples is significantly higher thanthat in 49 metastatic gastric carcinoma samples (Median, 60.6% vs.22.8%, P=0.044). According to the ROC curve for detection of gastriccarcinoma metastasis using GFRa1 methylation as a classifier, the areaunder the ROC curve (AUC) is 65.6% (P=0.004, FIG. 7). When the cutoffvalue is set at 16.4% (≤16.4% for GFRa1 demethylation-high and >16.4%for the demethylation-low), the demethylation-high rate in metastaticgastric carcinomas (35/49=71%) is significantly higher than innon-metastatic gastric carcinomas (24/49=49%) (P=0.038; sensitivity of71% and specificity of 51%). Kaplan-Meier analysis showed that GFRa1demethylation-high patients' overall survival is shorter than thedemethylation-low patients (5-year survival rate, 32.13 vs. 62.2%;log-rank test, P=0.001; multivariate analysis, P=002; FIG. 8).

In the validation cohort, the average peak-area proportion for themethylated GFRa1 in 47 non-metastatic cancer tissues is significantlyhigher than that in 73 metastatic cases (Median, 49.0% vs. 30.6%P<0.001). Using the same cutoff value (16.4%) used in the abovediscovery cohort, GFRa1 demethylation-high rate in the metastatic cases(46/73=63.1%) is significantly higher than that in non-metastatic cases(19/47=40.4%) (P=0.024; sensitivity of 63% and specificity of 60%).Kaplan-Meier analysis also shows that the demethylation-high patientshave a significant shorter overall survival than demethylation-lowpatients (5-year survival rate, 47.7% vs. 71.7%; log-rank test, P=0.015;multivariate analysis, P=0.025; FIG. 9).

EXAMPLE 3 Screening for Gastric Cancer Through Detection ofDemethylation of GFRa1 CpG Islands in Gastric Tissue UsingBisulfite-Sequencing

1. The same as the steps 1-5 in the Example 1.

2. The PCR products are cloned using the AT-Clone Kit and sequenced(FIG. 18).

3. Result: the same result is achieved as the Example 1.

EXAMPLE 4 Detection of Metastasis of Gastric Carcinomas and Patients'Survival Through Detection of Demethylation of GFRa1 CpG Islands UsingBisulfite-Sequencing

1. The same as the steps 1-2 in the Example 2.

2. The same as the step 2 in the Example 3.

3. Result: The same result is achieved as the Example 2.

EXAMPLE-5 Screening for Colon Cancer Through Detection of Demethylationof GFRa1 CpG Islands in Colon Tissue by Using of DHPLC

1. Subjects: colon mucosal biopsy samples from 21 non-cancer patientsand colon cancer and surgical margin samples from 97 patients;

2. The same as the steps 2-6 in the Example-1.

3. Result:

The average proportion of methylated-GFRa1 in the colon biopsies fromnon-cancer patients (Median, 64.1%) is significantly higher than coloncarcinomas (31.6%; P<0.001) and the surgical margin tissues (26.6%;P=001; FIG. 10). The area under the ROC curve is 74.1% according resultsof the methylation analysis using cancer tissues and control biopsiesfrom non-cancer patients (P<0.001; FIG. 11). When the cutoff value isset at 34.5% (≤34.5% for demethylation positive and >34.5% fordemethylation negative), GFRa1 demethylation positive rate in 21 controlbiopsies from non-cancer patients (7/21=33.3%) is significantly lowerthan that in 97 surgical margin sample (93/97=95.8%; P<0.001) and 97cancer samples (60/97=61.9%; P<0.001). Using GFRa1 demethylationpositive in the non-cancer biopsies or surgical margin samples as abiomarker for screening of colon cancer, the sensitivity and specificityis 95.8% and 66.7%, respectively.

EXAMPLE 6 Detection of Metastasis of Colon Carcinomas and Patients'Survival Through Detection of Demethylation of GFRa1 CpG Islands byUsing of DHPLC

1. Subjects: colon cancer tissues from 49 patients without metastasis,and metastatic colon cancer tissues from 48 control patients.Clinicopathologic and follow-up data are available for all of abovesubjects.

2. The same as the steps 2-6 in the Example 1.

3. Results: The average proportion of methylated-GFRa1 in 49non-metastatic colon cancers is significantly higher than that in 48metastatic cancers (Median, 45.6% vs. 25.0%, P=0.016). The area underthe ROC curve (AUC) is 62.6% (P=0.033; FIG. 12). When the cutoff valueis set at 27.6% (≤27.6% for GFRa1 demethylation-high and >27.6% for thedemethylation-low), the GFRa1 demethylation-high positive rate in 48metastatic cancers (33/48) is significant higher than in 49non-metastasis cancers (22/49) (68.8% vs. 44.9%, P=0.024; sensitivity of69% and specificity of 55%). Kaplan-Meier analysis showed that GFRa1demethylation-high cancer patients have a significant shorter overallsurvival than demethylation-low patients (3-year survival rate, 41.8%vs. 66.7%; log-rank test, P=0.019; multivariate analysis, P=0.031; FIG.13).

EXAMPLE 7 Screening for Colon Cancer Through Detection of Demethylationof GFRa1 CpG Islands in Colon Tissue Using the Probe-Based,Quantitative, Methylation-Specific PCR (MethyLight)

1. Subjects: The same as the step 1 in the Example 5.

2. DNA sample management: The same as the steps 2-3 in the Example 1.

3. According to the bisulfite-modified GFRa1 sense-strand sequence (SEQID NO.1) or antisense-strand sequence (SEQ ID NO.3), designing andsynthesizing forward and reverse PCR primers (SEQ ID NO.9 and SEQ IDNO.10) and sequence-specific fluorescent probe (SEQ ID NO.11) for theantisense-strand sequence, or designing and synthesizing forward andreverse PCR primers (SEQ ID NO.12, SEQ ID NO.13) and sequence-specificfluorescent probe (SEQ ID NO.14) for the sense-strand sequence.

4. Fluorescent probe-based, quantitative PCR amplification: the 158 bpbisulfite-modified template in both the methylated and demethylatedGFRa1 alleles is amplified using the fluorescence quantitative PCRamplification.

5. As reported in the literature (Widschwendter at al. Cancer Res 2004,64:3807-3813), the CpG island-free gene COL2A (but not limited to thegene), as the reference gene to normalize the amount of inputbisulfite-modified DNA template, is amplified using the correspondingthe primer set (SEQ ID NO. 15 and SEQ ID NO. 16) and the fluorescentprobe (TaqMan; sequence SEQ ID NO.17; 6FAM-Col2a^(probe)-BHQ1).

6. Calculating the proportion of methylated GFRa1 templates: based theCt values for GFRa1 and COL2A1, the relative copy number of methylatedGFRa1 is calculated using the formula [2^(−(Ct) ^(GFRA1) ^(−Ct) ^(COL2A)⁾].

7. Result: The average proportion of methylated GFRa1 in normal colonbiopsies (median, 46.8%) is significantly higher than in colon carcinomasamples (12.6%, P<0.01) or their corresponding surgical margin tissues(0.0005%, P<0.01). The area under ROC curve (AUC) is 69.7% (P<0.05).When the cutoff value is set at 1.3% (≤1.3% for GFRa1 demethylationpositive and >1.3% for the demethylation negative), the demethylationpositive rate in colon biopsies from non-cancer patients (8/20=40%) issignificantly lower than that in the surgical margin tissues (16/20=80%)and colon cancer tissues (97/97=100%, P<0.001). Sensitivity andspecificity for detection of colon cancer using the demethylation-highwas 80˜100% and 60%, respectively.

EXAMPLE 8 Detection of Metastasis of Colon Carcinomas andPatients'Survival Through Detection of Demethylation of GFRa1 CpGIslands Using the Probe-Based, Quantitative, Methylation-Specific PCR(MethyLight)

1. Subjects: The same as the step 1 in the Example 6.

2. DNA sample management and quantification of methylated GFRa1: Thesame as the steps 2-6 in the Example 7.

3. Setting the cutoff value. The average relative copy number ofmethylated GFRa1 in 49 non-metastatic colon cancers is significantlyhigher than that in 48 metastatic colon cancer samples (Median, 13.6%vs. 9.7%, P=0.047). The area under the ROC curve (AUC) is 61.7%(P=0.047; FIG. 14). When the cutoff value is set at 22.3% (≤22.3% forGFRa1 demethylation-high and >22.3% for the demethylation-low), theGFRa1 demethylation-high positive rate in 48 metastatic cancers (40/48)is significant higher than in 49 non-metastasis cancers (31/49) (83.3%vs. 63.3%, P0.038; sensitivity of 83% and specificity of 37%).Kaplan-Meier analysis showed that GFRa1 demethylation-high cancerpatients have a significant shorter overall survival thandemethylation-low patients (3-year survival rate, 46.5% vs. 69.2%;P=0.056; FIG. 15).

EXAMPLE 9 Screening for Cancers Through Detection of Demethylation ofGFRa1 CpG Islands in Plasma Free DNA Using the Probe-Based,Quantitative, Methylation-Specific PCR (MethyLight)

1. Subjects: The same as the Example 1 and Example 5; 0.3 ml ofanti-coagulated venous plasma from these fasting subjects is prepared.

2. According to the Blood DNA Extraction Kit (QIAGEN, Germany)Instruction Manual, free DNA sample is extracted from 0.3 ml of plasmasample from cancer and non-cancer control subjects.

3. The same as the step 3 in the Example 1.

4. The same as the steps 3-6 in the Example 7.

5. Result: The average proportion of methylated GFRa1 in the plasma freeDNA sample from non-cancer control subjects is significantly higher thanthat from colon cancer subjects (median, 66.5% vs. 10.2%, P<0.01). Thearea under the ROC curve (AUC) is 79.5% (P<0.01). When the cutoff valueis set at 2.3% (≤2.3% for GFRa1 demethylation positive and >2.3% for thedemethylation negative), GFRa1 demethylation positive rate in the plasmasamples from non-cancer subjects (4/20=20%) is significantly lower thanthat from colon cancer patients (97/97=100%/). The sensitivity andspecificity for screening of colon cancer using GFRa1 demethylation inplasma as a biomarker is 100% and 80%, respectively.

EXAMPLE 10 Detection of Metastasis of Hepatocellular Carcinomas andPatients' Survival Through Detection of Demethylation of GFRa1 CpGIslands by Using of DHPLC

1. Subjects: hepatocellular carcinoma samples from 37 patients,including 17 cases with metastasis/recurrence, 20 cases withoutmetastasis/recurrence. Clinicopathologic and follow-up data areavailable for all of these subjects.

2. DNA management and analysis of methylated GFRa1: The same as thesteps 2-6 in the Example 1.

3. Result: The average proportion of methylated GFRa1 in 20non-metastatic hepatocellular carcinomas is significantly higher thanthat in 17 metastatic cases (Median, 55.6% vs. 41.4%, P=0.065). The areaunder the ROC curve (AUC) is 68.4% (P=0.060; FIG. 16). When the cutoffvalue is set at 49.3% (≤49.3% for GFRa1 demethylation-high and >49.3%for the demethylation-low), GFRa1 demethylation-high positive rate ishigher in the metastatic cancers (12/17=70.5%) than non-metastaticcancers (11/20=55.0%) (P=0.067). The sensitivity and specificity ofdetection of liver cancer metastasis using GFRa1 demethylation-high as abiomarker is 70% and 45%, respectively. Kaplan-Meier analysis showedthat patients with GFRa1 demethylation-high liver cancer have a shorteroverall survival than those with GFRa1 demethylation-low liver cancers(FIG. 17).

What is claimed is:
 1. A method of detection of metastasis of cancersand postoperative survival in a human having gastric cancer, coloncancer or hepatocellular carcinoma, characterized in that said methodcomprises the following steps: a) extracting gastric, colon or livertissue DNA or plasma free DNA samples from a certain number of patientswith metastatic cancer and patients with non-metastatic cancer; b)detecting and calculating the proportion of methylated or demethylatedGFRa1 CpG islands in the promoter region of GFRa1 of said DNA from a);setting a cutoff value for detection of metastasis of cancers andpatients' postoperative survival using-the proportion of methylated ordemethylated GFRa1, wherein the cutoff value of methylated ordemethylated GFRa1 is calculated by using a Receiver OperatingCharacteristic (ROC) curve; c) extracting gastric, colon or liver tissueDNA or plasma free DNA samples from human testing subjects, detectingand calculating the proportion of methylated or demethylated GFRa1 CpGislands in the DNA samples; d) comparing the proportion of methylated ordemethylated GFRa1 determined in the step c) with the cutoff value setin the step b); and e) detecting the metastasis of cancers and patients'postoperative survival based on the detection of a less than or equalproportion of methylated GFRa1 CpG islands determined in step c) ascompared to the cutoff value set in the step b), or based on thedetection of a higher than or equal proportion of demethylated GFRa1 CpGislands determined in step c) as compared to the cutoff value set in thestep b), wherein the proportion of the methylated or demethylatedGFRa1CpGs islands is determined and calculated by DHPLC orbisulfate-sequencing using following primer sets: i) the primer pair setforth in SEQ ID NO: 5 and SEQ ID NO: 6; or ii) the primer pair set forthin SEQ ID NO: 7 and SEQ ID NO:
 8. 2. The method according to claim 1,wherein the proportion of the methylated or demethylated GFRa1 allelesdescribed in the steps b) and c) is determined and calculated by thefollowing assays: chemical modification of unmethylated cytosine;designing and synthesizing primer sets for PCR amplified of themethylated or demethylated GFRa1 CpG islands according to their modifiedsequences; determining and calculating the proportion of methylated ordemethylated GFRa1 CpG islands with quantitative analysis ofmethylation.
 3. The method according to claim 2, wherein the modifiedsequences of GFRa1CpG islands described in the steps b) and c) are asshown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.